The present invention pertains in general to antigens associated with tumors and uses therefor, and in particular to regression-associated antigens (RAAs), to recombinant methods for producing RAAs in E. coli, to preparations containing RAAs and to uses for such antigens and preparations.
One approach to diagnosis and treatment of cancers involves the development of polyclonal and monoclonal antibodies against tumor-associated antigens. In almost all reported cases, the immunogens used to obtain antibodies directed against tumor cell components are intact tumor cells or are membrane proteins obtained from the cells.
Early work in this field involved the identification of onco-fetal antigens and blood group antigens [Springer, Science, 224, 1198 (1984)] which are expressed by malignant cells and shed into the bloodstream in some instances. Antigens associated with tumor cells may be identified by immunoblotting methods. Du Bois et al., J. Immunol. Methods, 63, 7 (1983).
In particular, monoclonal antibodies reactive with the surface of human breast carcinoma cells may be generated and characterized using membrane-enriched fractions of metastatic carcinoma lesions. Schlom et al., Cancer, 54 (11 Suppl.), 2777-2794 (1984). One monoclonal antibody is reported to react with a 220,000 to 400,000 dalton high molecular weight glycoprotein complex found in 50% of human mammary carcinomas and 80% of human colon carcinomas. Scholm et al., supra. Mouse monoclonal antibody L6 is reported to recognize a ganglioside antigen that is of particular interest because it is expressed at the surface of cells from most human carcinomas of lung, breast, colon and ovary, while it is present in only trace amounts at the surface of normal cells. Hellstrom et al., Cancer Res., 46, 3917-3923 (1986).
A number of other monoclonal antibodies reactive with tumor-associated antigens on the surfaces of other human cancer cells, including ovarian, pancreatic and intestinal malignancies, may thus be obtained. Proceedings of the UCLA Symposium on Monoclonal Antibodies and Cancer Therapy, Reisfeld et al., eds., Alan R. Liss, Inc., New York, 3-74, 97-109 and 149-164 (1985).
Monoclonal and polyclonal anti-tumor cell antibodies described to date are directed against determinants of human tumor cell antigens which may elicit an immune response in test animals chosen for the production of tumor-specific antibodies. It is not known whether patients harboring tumors or treated with specific and/or non-specific immune stimulants produce antibodies against these antigenic determinants. Therefore, the relevance of such antibodies in mediating regression of tumors in patients is unclear. Passive transfer of such antibodies generated in animals into patients has met with limited success. Lowder et al., Western J. Med., 143, 810 (1985).
A clinical approach toward active immunotherapy of tumors involves a generalized stimulation of the patient's own immune system using non-specific stimulants such as components of the walls of two bacterial cells, Mycobacterium bovis (BCG strain) and Corynebacterium parvum, or "detoxified" bacterial endotoxin. In parallel, biological response modifiers such as interleukin-2 may be used to induce activation of the immune system and cause tumor cell destruction. Mule et al., J. Immunol., 135, 646 (1985); and Rosenberg et al., New Engl. J. Med., 313, 1485 (1985).
In an approach called active specific immunotherapy, immunization of cancer patients may be attempted with preparations derived from allogeneic tumor cells (tumor cells obtained from a histopathologically similar tumor of a different patient). This may specifically stimulate the patient's own immune system to possibly unique antigenic structures present on a particular malignant cell type, and may thus induce tumor regression. Lachman et al. Br. J. Cancer, 51, 415-417 (1985); and Wallack et al., Surgery, 96, 791-800 (1984). Active specific immunotherapy may also be attempted by systematically injecting autologous (autochthonous) tumor cells (i.e., cells derived from the tumor mass of the same patient) intradermally or subcutaneously. Laucius et al., Cancer, 40, 2091 (1977).
Various preparations of autologous tumor cells or of allogeneic tumor cell lines have been used in active specific immunotherapy. Key et al., Adv. Immun. Cancer Ther., 1, 195-219 (1985); Weisenburger et al., J. Biol. Response Mod., 1, 57-66 (1982); and Kan-Mitchell et al., Proceedings of the UCLA Symposium on Monoclonal Antibodies and Cancer Therapy, supra, 523-536. The preparations are generally treated with irradiation, mechanical disruption, or freeze-thaw cycles to render the tumor cells non-viable. They are then used as immunogens, with or without an adjuvant, and are administered by a variety of routes (such as intradermal, subcutaneous, intramuscular or intralymphatic) for the purpose of immunizing cancer patients. Relatively little toxicity has been reported with these preparations, and encouraging clinical responses have been obtained in significant numbers of advanced cancer patients.
Serum samples may be obtained from patients with documented malignancies that are in the state of tumor progression from the primary site of origin to other locations in the body (metastasis) or by a demonstrable growth of the primary tumor mass. The patients may then be subjected to intralymphatic immunotherapy as described in Juillard et al., Bull. Cancer, 66, 217 (1979), using infusions of tumor cells obtained from their own tumors or cultured tumor-derived cells established from malignancies of the same type as the patient in question as described in Juillard et al., Cancer, 41, 2215 (1978); and in Weisenburger et al., J. Biol. Response Mod., 1, 57 (1982). The amount of neoplastic cells used for immunization and methods of their processing including washing and irradiation prior to administration into patients is reported. Juillard et al., Cancer, 41, 2215-2225 (1978); and Bubbers et al., Bull. Cancer, 68, 332-337 (1981). However, such procedures do not identify RAAs or disclose recombinant production of RAAs.
One major limitation of attempts at active specific immunotherapy is the undefined nature of the tumor cell preparations (generally intact irradiated cell suspensions or mechanically disrupted lysates of cells). Cells from autologous tumor cells grown in tissue culture or continuously passaged tumor cell lines may undergo significant changes in their phenotypes during growth in laboratory culture. Reagents or tests to standardize the preparations for their expected potency have not been available. Membrane proteins may be shed from intact irradiated cells and proteins in cell lysates may be degraded by proteolytic enzymes. Different preparations used in attempts at active specific immunotherapy may, therefore, have variable efficacies although similar processes and cell types are utilized. Furthermore, the means of monitoring the immune response of individual patients is not available for tailoring the immunization dose and the immunization schedule for optimal clinical outcome. In addition, it is often the case that autologous tumor cell preparations are not practical because of a lack of an adequate amount of tumor from the patient to be treated.
The results of clinical studies with autologous tumor cell vaccines are encouraging when a potent adjuvant such as BCG is used along with the tumor cell suspension. Hoover et al., Cancer Res., 44, 1671-1676 (1984). Some patients immunized through different routes with allogeneic cells, with or without adjuvants, have shown significant, often dramatic, clinical responses. Weisenburger, J. Biol. Response Mod., 1, 57-66 (1982); Mitchell, in Proceedings of the UCLA Symposium on Monoclonal Antibodies and Cancer Therapy, supra, 495-504. Certain key antigen components of tumor cells may be able to elicit protective/regressor antibodies in humans.
Results from a number of animal models support the use of tumor cell components in active specific immunization to induce tumor regression [Key et al., J. Biol. Response Mod., 3, 359-365 (1984); and Srivastava et al., Proc. Nat'l Acad. Sci. (USA), 83, 3407-3411 (1986)]. Neoplasms induced in mice by polycyclic aromatic hydrocarbons such as 3-methylcholoanthrene express individually distinct tumor-associated transplantation antigens. These antigens are immunogenic in their syngeneic hosts and provide transplantation immunity only against their respective tumors and not against independent tumors induced by the same or a different carcinogen or against tumors of viral origin. Transplantation immunity in mice may be elicited by prior growth and removal of tumor transplants or by immunization with irradiated tumor cells, tumor cell membranes or solubilized antigen preparations.
A monoclonal antibody designated PF/2A is a product of standard monoclonal antibody production techniques involving injection into mice of cells of a human squamous lung carcinoma cell line. PF/2A antibody is reported to react with breast carcinoma, colon carcinoma, gastric carcinoma, tumors of ectodermal origin and squamous lung cell carcinomas as well as a 24 kilodalton ("kd") polypeptide extracted from squamous lung cell carcinoma cells [Fernsten et al., Cancer Res., 46, 2970-2977 (June, 1986)]. Monoclonal antibody PF/2A is also reported to immunoprecipitate a 46 kd polypeptide extracted from human cell lines infected with Mycoplasma hyorhinis (M. hyorhinis), and stains, but to not precipitate, a 24 kd component derived from M. hyorhinis [Fernsten et al., Infect. Immun., 55, 1680-1685 (July, 1987)]. However, no association with regression of tumors is shown or suggested for this antibody or antigens reactive with the antibody by Fernsten et al.
In Gussack et al., Cancer, 62, 57-64 (1988), it is reported that most primary human carcinomas uniformly express an oncofetal epitope which is reported not to have been demonstrated previously in established human carcinoma cell lines. It is further reported that several low-passage cell lines of human squamous cell carcinoma ("SCC") from head and neck tumors are derived, characterized and examined for expression of a 44 kd polypeptide oncofetal antigen ("OFA") at the cell surface. These new cell lines and two long-term, established SCC lines (FaDu and Detroit 562) are reported to displayed OFA at the cell surface, as determined by flow cytometry using a monoclonal antibody. It is proposed that the expression of a 44 kd OFA is a common feature of human SCC, and that this marker may prove useful in the detection and treatment of these tumors. Nevertheless, no indication is given in Gussack et al. that the proposed 44 kd OFA is a regression-associated antigen.
In Hollinshead et al., Cancer, 60, 1249-1262 (1987), the 10-year cumulative experiences of five year survivals of patients entered into a successful phase II specific active tumor-associated antigen ("TAA") immunotherapy trial, a successful phase III specific active immunotherapy trial A and of patients in an unsuccessful specific active immunotherapy trial B are reported. The TAAs used are reported to be lung tumor cell membrane components which produce cell mediated components which produce cell-mediated immunity as measured in vivo and in vitro. In addition, monoclonal antibody-derived epitope enzyme immunoassays are reported using a 37 kd lung squamous cell TAA to monitor specific, early antibody rises in the bloodstream. However, no relationship of the TAAs to regression-associated antibodies is reported.
In Young et al., Proc. Natl. Acad. Sci. (USA), 85, 4267-4270 (1988), it is reported that, to understand the immune response to infection by tuberculosis (M. tuberculosis) and leprosy (M. leprae) bacilli and to develop improved vaccines, an investigation of the nature of antigens that are involved in humoral and cell-mediated immunity is discussed. Five studied immunodominant protein antigens (three from M. leprae, 71 kd, 65 kd and 18 kd; and two from M. tuberculosis, 70 kd and 65 kd) are reported to be homologs of stress proteins. It is indicated that this finding and observations with other pathogens suggested that infectious agents may respond to the host environment by producing stress proteins and that these proteins may be important immune targets, so that it is postulated in Young et al. that abundant and highly conserved stress proteins may have "immunoprophylactic" potential for a broad spectrum of human pathogens. Nevertheless, no relationship to tumor regression is disclosed.
In Jessup et al., Arch. Surg., 122, 1435-1439 (1987), the antibody response of patients is reported to be used to characterize autoantigens in human colorectal carcinoma. Primary and metastatic carcinomas with paired normal tissues are reported to be extracted and transferred onto nitrocellulose membranes by the Western transfer technique which are incubated with the serum of the patient from whom the tumor was derived. Autoantigens are reported to be identified by indirect immunoperoxidase staining. All tumors are reported to contain at least one autoantigen. Six tumor-associated autoantigens (reported to have molecular weights of 26 kd, 29 kd, 32 kd, 38 kd, 41 kd and 58 kd are reported to be identified by antibodies in 25% or more of the sera. Eleven metastases are reported to express a 41 kd autoantigen present in only a third of the extracts of normal liver or lung. Thus, the number of dominant polypeptide autoantigens in colorectal carcinoma is reported to be restricted to six molecules. These autoantigens may be organ-associated antigens that are expressed by neoplastic cells. The 41 kd autoantigen is reported to be a potential marker for metastases. A generic vaccine is reported to appear to be feasible for colorectal carcinoma since the number of dominant antigens is limited. However, no relationship of the autoantigens to regression is reported although it is suggested that autoantibodies to cytoplasmic antigens may be important for the survival of the patient.
In Law et al., Cancer Res., 47, 5841-5845 (1987) is reported the characterization of a 65 kd tumor rejection antigen obtained from a murine malignant melanoma. Greater than 95% inhibition of primary tumor growth in a mouse system is reported for the use of irradiated 591 murine malignant melanoma cells expressing the 65 kd antigen, but the extracted and purified 65 kd antigen from 591 cells is reported to be in effective in inhibiting primary tumor growth in a mouse system. Although a 65 kd melanoma specific tumor rejection antigen from another murine malignant melanoma cell line (B16) is reported to effective in inhibiting tumor growth, no relationship of the reported antigen to regression-associated antibodies is reported.
A knowledge of the antibody response associated with human tumor regression following active specific immunotherapy and identification of subcellular components involved in eliciting such specific antibodies should lead to the development of improved active specific immunogens for cancer immunotherapy. Thus, it is desirable to develop: (i) preparations which will be more enriched in the relevant specific immunogens; (ii) reagents to screen better cell sources and quantitate the immunogens in preparations derived from these cells such that different preparations may be meaningfully standardized; and (iii) assay methods to monitor patients' specific immune response to these immunogens, thereby providing the physician an ability to adjust the treatment protocol in order to produce a better clinical outcome.